Additional file 1: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Questionnaire completed by families recruited to the study. (PDF 28 kb

Additional file 5: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Figure S2. Procrustes analysis of raw and rarefied datasets. The rarefied dataset was subsampled at a threshold of 1499 reads per sample. The raw dataset excluded samples below this depth. P-values are non-parametric and are based on 999 Monte Carlo simulations. (PNG 174 kb

Vitamin D deficiency increases bacterial load in the lungs of male mice with allergic airway disease.

<p>Male and female offspring born to vitamin D-replete or -deficient mothers and maintained on the vitamin D-replete or null diets (respectively), or vitamin D-deficient offspring switched to a vitamin D-replete diet from 8 weeks of age (VitD- to VitD+), were sensitised at 12 weeks of age intraperitoneally with 1 µg ovalbumin (OVA) (0.2 mg Aluminium hydroxide (Alum)). Mice were boosted with the same OVA/Alum dose two weeks after the initial sensitisation, and one week later their airways challenged with aerosolised OVA (1% in saline). Shown are bacterial DNA levels induced relative to levels in vitamin D-replete, sex-matched mice as measured in the lungs of mice using a PCR with redundant primers for detection of bacterial 16 S rRNA gene 24 h after the OVA aerosol (mean + SEM for ≥12 mice/treatment from three independent experiments, *p<0.05 between groups, #p<0.05 relative to levels in vitamin D-replete mice).</p

Additional file 9: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Figure S5. Procrustes analysis of left and right ear samples. The dataset includes both MEF and MER samples in left/right ear pairs from the same child. Samples with less than 1499 reads are excluded. The p-value is non-parametric and is based on 999 Monte Carlo simulations. (PNG 91 kb

Additional file 3: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Table S1. Full taxonomy of all OTUs identified in this study. Taxonomy is from the SILVA database, v123 for QIIME. Taxonomy assigned to OTUs by UCLUST v1.2.22q within QIIME v1.9.1 (assign_taxonomy.py). (XLSX 13 kb

Additional file 6: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Figure S3. Beta diversity PCoA in the nasopharynx of cases and controls, sorted by other covariates. Case and control nasopharyngeal samples shown in Fig. 3 are coloured by other covariates. NA refers to samples where the covariate was not applicable or was missing (not given or recorded “unknown”) and the number represents individual samples. (PDF 564 kb

Additional file 2: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Figure S1. Diagrammatic overview of the 16S rRNA gene data analysis pipeline. Names of the software or tools used are in red. The SILVA database replaced the default taxonomy database in QIIME (GreenGenes) as GreenGenes 13_8 version does not discriminate between Alloiococcus and Dolosigranulum. (PDF 366 kb

Additional file 10: of A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera

Figure S6. Procrustes analysis of MEF and MER samples. The dataset includes pairs of MEF and MER samples from the same ear of the same child. Samples with less than 1499 reads are excluded. The p-value is non-parametric and is based on 999 Monte Carlo simulations. (PNG 80 kb

Vitamin D deficiency does not affect the immunogenicity of dendritic cells or the numbers of regulatory T cells in the airway-draining lymph nodes.

<p>Eight week-old male and female offspring born to vitamin D-replete or -deficient mothers and maintained on the vitamin D-containing or -null diets (respectively) were sensitized intraperitoneally with 1 µg ovalbumin (OVA) (0.2 mg Aluminium hydroxide (Alum)). Mice were boosted with the same OVA/Alum dose two weeks after the initial sensitization, and one week later their airways challenged with aerosolised OVA (1% in saline). Cells were assessed twenty-four h after OVA challenge. In (A), the capacity of CD11c+ cells isolated from the ADLN to process and present antigen to co-cultured CD4+ T cells from DO11.10 mice was determined by [³H]-thymidine incorporation for the final 24 h of 96 h co-culture with 1 µg/ml OVA peptide. Results are shown of a representative of 3 independent experiments (mean±SEM) for CD11c+ cells cultured at various ratios with CD4+ cells. In (B), the FACS gating strategy used to determine the percentage of ClassII+CD11c+CD8− cells in ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (C), the FACS gating strategy used to determine the percentage of CD3+CD4+ T cells and CD3+CD4+CD25+Foxp3+ and CD3+CD4+CD25+Foxp3− cells in the ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (D), the expression of CCL5, CCL11, CCL19, CCL20, CXCL1 and IL-18RA mRNAs in CD11c+ cells isolated from pooled ADLN of ten mice per treatment with results shown from 3 independent experiments (mean+SEM). (*p<0.05).</p

Multiple ovalbumin aerosols overcome the capacity of physiological vitamin D to modulate airway inflammation.

<p>Eight week-old male and female offspring born to vitamin D-replete or -deficient mothers and maintained on the vitamin D-containing or null diets (respectively) were sensitized intraperitoneally with 1 µg ovalbumin (OVA) (0.2 mg Aluminium hydroxide (Alum)). Mice were boosted with the same OVA/Alum dose two weeks after the initial sensitization, and one week later their airways challenged with aerosolised OVA (1% in saline) on three consecutive days. The total number of bronchoalveolar lavage fluid (BALF) cells, and the number of eosinophils and neutrophils 24 h after the final OVA challenge are shown along with IL-5 levels. Data is shown as mean+SEM for 10 mice/treatment combined from two independent experiments.</p